DISCUSIÓN
4. Expresión de MCT8 en el SNC durante el desarrollo fetal y la infancia
3.1 Particle size distribution and ǮǮ-potential of lipid vesicular systems 207
In this study, different lipid vesicular systems, including conventional liposomes, invasomes 208
and ethosomes, were prepared and characterized in order to evaluate their capability to improve skin 209
delivery of two model drugs, hydrophilic model drug CF and lipophilic model drug mTHPC. The 210
compositions of these different lipid vesicular systems are reported in Table 1 and their 211
corresponding results of particle size distribution and Ǯ-potential are summarized in Table 2.
212
Conventional liposomes showed the largest mean vesicle size of 121.7±0.8 nm (n=3) and 213
114.6±2.0 nm (n=3) for CF and mTHPC, respectively. Invasomes (115.3±1.4 (n=3) and 109.9±0.2 nm 214
(n=3) in the case of invasomes containing CF and mTHPC, respectively) and ethosomes (81.6±5.8 215
(n=3) and 77.8±0.5nm (n=3) in the case of ethosomes containing CF and mTHPC, respectively) had 216
significantly (P<0.01) lower mean vesicle size relative to corresponding conventional liposomes. In 217
the case of ethosomes, the presence of high concentration of ethanol (45%, v/v) could be the 218
reasonable explanation about the reduced particle size of ethosomes in comparison with 219
conventional liposomes. Ethanol could probably reduce the membrane thickness due to the 220
formation of a phase with interpenetrating hydrocarbon chains (Barry and Gawrisch, 1994; Dubey 221
et al., 2007). Furthermore, the addition of ethanol in phospholipid vesicles imparts negative charge 222
to the formulation and this modification of net charge of the system confers lipid vesicles some 223
degree of stearic stabilization and in turn lead to decrease in mean vesicle size (Jain et al., 2007). All 224
of these mechanisms suggested that ethanol possesses some condensing ability for lipid vesicles.
225
While, in the case of invasomes, the incorporation of 10% of ethanol could also reduce the particle 226
size distribution with the same mechanism. Moreover, another important component, 227
lysophosphatidylcholine (LPC) which was one of the components of NAT 8539 works as a 228
surfactant creating a high positive curvature in membranes (Fuller and Rand, 2001). However, the 229
inclusion of 1% (w/v) terpenes mixture could lead to the increase of particle size distribution 230
(Dragicevic-Curic et al., 2008). Regarding the polydispersity index (PDI), all the lipid vesicular 231
systems showed low values (PDI<0.2), indicating that all of them were highly homogeneous 232
suspensions.
233
The ζ-potential is related to the charge on the surface of the vesicle which influences both 234
vesicular properties such as stability, as well as skin–vesicle interactions. Conventional liposomes 235
containing CF or mTHPC prepared in this study were found to possess a ζ-potential of -12.3±0.7 236
mV (n=3) and -6.2±1.4 mV (n=3), respectively. Invasomes containing CF or mTHPC exhibited a 237
negative ζ-Potential of -41.1±1.5 mV (n=3) and -39.4±1.2 mV (n=3), respectively. This result is 238
unreasonable agreement with the recent research data on invasomes containing mTHPC from our 239
department (Dragicevic-Curic et al., 2008, 2009). Ethosomes containing CF or mTHPC also 240
showed a negative ζ-Potential of -79.7±1.4 mV (n=3)and -84.1±1.4 mV (n=3), respectively. In the 241
case of ethosomes, incorporation of DPPG (Samad et al., 2007), is expected to produce highly 242
negatively charged vesicles. The effect of surface charge of liposomes on the drug penetration has 243
not been fully understood so far. Some researchers support the theory that the positive charges on 244
the surface of liposomes could bind to negative charges of the SC enhancing thereby the drug 245
penetration/permeation through the skin (Katahira et al., 1999; Song and Kim, 2006). However, 246
other studies found that permeation of drugs through the skin is promoted by negatively charged 247
vesicles (Ogiso et al., 2001; Sinico et al., 2005). According to the best of our knowledge, the surface 248
charge of the vesicles will not only play a role on the interaction between skin and vesicles but also 249
might contribute to the drug release from the vesicles. The drug release from vesicles in the stratum 250
corneum is an important step which will affect transdermal flux (Honeywell-Nguyen and Bouwstra, 251
2003). The rate and amount of released drug is a balance between two factors: (1) drug affinity to 252
vesicles, and (2) drug solubility in lipids of the stratum corneum (Honeywell-Nguyen and Bouwstra, 253
2003). In our previous study, another amphiphilic model drug, ferulic acid (FA), was used to 254
investigate the effect of surface charge of liposomes on the drug skin permeation. This study (Chen 255
et al., 2010) revealed that the flux of FA from negatively charged ethosomes is somewhat higher 256
than from positively charged ethosomes, but without any significant difference. Since the pKa1 of 257
FA is 4.52 (Erdemgil et al., 2007), it is negatively charged at pH 7.4. Therefore we suggested that FA 258
could be retained in the positively charged vesicles, which in turn influences the skin penetration or 259
permeation. In the case of CF, since it is also negatively charged at pH 7.4 (CF has a pKa of 6.3 260
(Nicole et al., 1989)), we preferred to prepare the negatively charged lipid vesicles containing CF 261
for comparison.. For the effect of positively charged liposomes and neutralized liposomes on the 262
skin penetration and deposition of CF and mTHPC is investigated in a forthcoming study.
263
Table 2 should be inserted here 264
3.2 Morphology of different lipid vesicles 265
Cryo-Transmission electron microscopy was used to visualize vesicles, and to study the shape 266
and lamellarity of different lipid vesicles containing CF (Fig.3A) or mTHPC (Fig.3B). From the 267
results, no matter if CF or mTHPC was encapsulated, the lipid vesicles had almost same shapes and 268
structures. The vesicles of the conventional liposomes seemed to be unilamellar (Fig. 3a, b, g and h, 269
black light arrows) and rarely bilamellar (Fig. 3a, b, g and h, black thick arrows), almost spherical 270
and oval in shape, and some detected oligolamellar vesicles (Fig. 3a, b, g and h, white arrows). In 271
the case of invasomes, the vesicles seemed to be almost unilamellar (Fig. 3c, d, i and j, black light 272
arrow) and bilamellar (Fig. 3c, d, I and j black thick arrow). Regarding ethosomes, the vesicles 273
appeared to be homogenously unilamellar (Fig. 3e, f, k and l, black light arrow).
274
3.3 In vitro skin penetration and skin deposition studies 275
3.3.1 Finite dose application for CF and mTHPC in vitro study 276
Penetration and deposition data across full-thickness human skin with non-occlusive 277
application of a finite dose (10µL/cm2) for CF or mTHPC after 12 hr by a range of formulation 278
vehicles are shown in Table 3 and Table 4, respectively, with their distribution in different skin 279
layers profiles shown in Figure 4 and Figure 5, respectively.
280
Table 3 should be inserted here 281
Table 4 should be inserted here 282
In the case of CF, the highest CF accumulation from all the test formulations (Table 3) was 283
found in the SC superficial layer (Stratum Corneum tape stripping layer Nr.1-5, SC L1-5) where 284
ethosomes containing CF (CF-ETS) and hydroethanolic solution containing CF (the mixture 285
solution of ethanol and PBS pH 7.4 (9:11, v/v), CF-HE) significantly enhanced CF accumulation in 286
comparison with PBS (pH7.4) solution containing CF (CF-PBS) group (by a factor of 8.9 and 8.1, 287
respectively, p < 0.01). Conventional liposomes containing CF (CF-CL) slightly improved CF 288
accumulation in SC L1-5 in comparison with CF-PBS (by a factor of 1.5; p < 0.05).CF 289
accumulations in SC deep layer (Stratum Corneum tape stripping layer Nr. 6-10, SC L6-10) and in 290
epidermis were all improved when using all the lipid vesicular systems as well as CF-HE in 291
comparison with CF-PBS, but with different magnitude . In the case of CF accumulation in SC 292
L6-10, CF-ETS showed the highest potential, followed by CF-HE > CF-INS > CF-CL> CF-PBS.
293
For CF accumulation in epidermis, both CF-ETS and CF-INS showed the highest CF accumulation 294
in epidermis, followed by CF-CL > CF-HE > CF-PBS. CF accumulation in dermis was only 295
significantly improved by CF-ETS in comparison with CF-PBS (by a factor of 2.2) and other 296
formulations didn’t show any significant enhancement. Furthermore, no permeation of CF through 297
the full thickness human skin was detected with this application condition from all the test 298
formulation vehicles.
299
Figure 4 should be inserted here 300
In the case of mTHPC, the highest mTHPC accumulation from all the test formulations (Table 301
4) was also found in the SC L1-5 where mTHPC-HE (hydroethanolic solution of ethanol and PBS 302
pH 7.4 (6:4, v/v) containing mTHPC) showed the highest mTHPC accumulation, followed by 303
mTHPC-CL> mTHPC-ET> mTHPC-ETS> mTHPC-INS. Regarding mTHPC accumulation in SC 304
L6-10 and in epidermis, mTHPC-HE also showed the highest potential, but followed by different 305
orders: mTHPC-CL> mTHPC-ETѩmTHPC-ETS≥ mTHPC-INS and mTHPC-ETS> mTHPC-ETѩ 306
mTHPC-INS≥ mTHPC-CL, respectively. The comparison of these five formulations showed that 307
significant differences existed between mTHPC-HE and others (p< 0.01) regarding the mTHPC 308
accumulation in these three different skin layers. However, the permeation of mTHPC through the 309
full thickness human skin and mTHPC accumulation in dermis were not detected with this 310
application condition for all test formulations.
311
Figure 5 should be inserted here 312
As can be seen, significant differences can be found between lipid vesicular systems containing 313
CF and mTHPC with respect to drug skin distribution profile of CF or mTHPC. In the case of 314
mTHPC, most of mTHPC can be found in SC superficial layers (SC L1-5) (Figure 5 and Table 4).
315
The percentage of mTHPC present in SC L1-5 was 94.0%, 84.8% and 92.6% of the total mTHPC 316
delivered for conventional liposomes (mTHPC-CL), invasomes (mTHPC-INS) and ethosomes 317
(mTHPC-ETS), respectively. While, in the case of CF, even though the highest drug accumulation 318
was also found in SC L 1-5, the drug skin distribution differed a lot according to the lipid vesicles 319
applied (Figure 4 and Table 3). The percentage of CF present in SC L1-5 was 51.4%, 30.9% and 320
54.1% of the total CF delivered for conventional liposomes (CF-CL), invasomes (CF-INS) and 321
ethosomes (CF-ETS), respectively.
322
In order to explain this drug skin distribution difference, it is necessary to consider possible 323
mechanisms of action of different lipid vesicular systems. Conventional liposomes were expected to 324
be effective at delivering drugs into the upper layers of the skin. It is agreed in the recent literature 325
that in most cases conventional liposomes are not penetrating the skin but remain confined to upper 326
layers of the SC or form a deposit on the surface of the skin (Tanner and Marks, 2008). The 327
penetration properties of conventional liposomes may fall into one of two possible categories, 328
including the penetration enhancing effect and vesicle adsorption to and/or fusion with the SC (El 329
Maghraby et al., 2006). The first possible mode of action that was described firstly in 1987 (Kato et 330
al., 1987) and supported by others (Hofland et al., 1995; Kirjavainen et al., 1999; Zellmer et al., 331
1995). This mode suggests that liposomal lipids may act as penetration enhancers, thereby 332
loosening the lipid structure of the SC and promoting an impaired barrier function (Kirjavainen et al., 333
1999). The second possible mode for conventional liposomes is adsorption to and/or fusion with the 334
SC (Abraham and Downing, 1990; Hofland et al., 1995; Kirjavainen et al., 1996), suggesting that 335
the liposomal lipids penetrate into the SC by adhering onto the surface of the skin and subsequently 336
destabilizing and fusing or mixing with the lipid matrix (Kirjavainen et al., 1996). However, the 337
collapse of vesicles on skin surface may form an additional barrier, reducing the permeation of 338
hydrophilic molecules encapsulated in the vesicular aqueous core (Elsayed et al., 2007b).
339
Regarding invasomes (belonging to the class of deformable liposomes) due to the presence of 340
lysophosphatidylcholine (LPC) and ethanol and terpenes (Dragicevic-Curic et al., 2008; Verma and 341
Fahr, 2004). Hence, there are two possible mechanisms responsible for its enhanced skin drug 342
delivery (Dragicevic-Curic et al., 2008). First, invasomes may act as drug carrier systems by which 343
intact vesicles can enter the SC carrying vesicle-bound drug into or across the skin. Second, 344
invasomes may act as penetration enhancers, whereby the vesicle lipid bilayers interact with the SC 345
and subsequently modify the intercellular lipid lamellae. It may also possible that one of the two 346
mechanisms might predominate according to the physicochemical properties of the drug considered 347
(Elsayed et al., 2007b).
348
The enhancing effect of ethosomes could be attributed to the synergistic mechanism between 349
ethanol, lipid vesicles and skin lipids (Dayan and Touitou, 2000; Elsayed et al., 2006; Touitou et al., 350
2000). Firstly, ethanol is a well-known permeation enhancer. The penetration enhancing effect of 351
ethanol can be attributed to two effects: (a) ‘Push effect’: increased thermodynamic activity due to 352
evaporation of ethanol and improved solubility of solute in this study; (b) ‘Pull effect’: ethanol can 353
interact with intercellular lipid molecules in the polar head group region, thereby increasing their 354
fluidity and decreasing the density of the lipid multilayer, which results in an increase in membrane 355
permeability. Ethanol is also supposed to extract the SC lipids (Bach and Lippold, 1998) lowering 356
thereby the barrier function of the SC. In addition, ethanol imparts fluidity to the vesicle's bilayers, 357
which in turn facilitates vesicles skin permeation. Furthermore, ethanol can act as ǎblending” agent 358
for lipid vesicles with increasing their distribution in various skin layers (Panchagnula et al., 2005).
359
The ethanol effects can be followed by the interaction between ethosomal vesicles and the skin. The 360
ethosomal vesicles may also behave as deformable liposomes and can interact with the skin barrier 361
to “forge” penetration or permeation pathways by itself in the highly organized SC and finally 362
release drug at various points along the penetration pathway as well as in deep skin layers (Elsayed 363
et al., 2007b; Godin and Touitou, 2003).
364
Moreover, the different molecular mechanisms by which the diffusion through the stratum 365
corneum of hydrophilic molecule (CF) and lipophilic molecule (mTHPC) should be also taken into 366
account, because drug skin penetration and deposition via lipid vesicular systems involves several 367
processes, including interaction between SC and lipid vesicles, partitioning of the drug from its lipid 368
vesicular system to the skin and the following drug diffusion in the skin. Drugs are considered to 369
penetrate through the skin by one of three pathways: the polar, non-polar, or polar/non-polar route 370
depending on their physicochemical properties, in which logPo/w of drugs is thought to be the key 371
factor (Verma and Fahr, 2004). The logPo/w value, which is a measure of how well a substance 372
partitions between a lipid and water, determines the route of drug penetration through the skin.
373
Temoporfin (mTHPC), which is highly lipophilic, is expected to penetrate the skin by non-polar 374
pathways, whereas CF, which is hydrophilic, should utilize the polar pathways. The intrinsic 375
permeability of both hydrophilic and lipophilic penetrants is governed by the composition of the 376
skin, with the former limited by their partitioning into the lipophilic SC and the latter, by 377
partitioning from the SC into the less lipophilic epidermis(Nicoli et al., 2008; Zhang et al., 2010). 378
Consequently, the logPo/w value of drug molecule has an effect on the enhancement efficacy of 379
penetration enhancers. Hydrophilic molecules such as CF, owing to their low partition coefficient 380
and high hydrogen-bonding potential, would show a dramatic increase in permeation with suitable 381
enhancers, however, lipophilic molecules which move with relative ease through the SC do not have 382
the same opportunity to act as indicators of enhancement (Barry and Bennett, 1987; Verma and Fahr, 383
2004; Zhang et al., 2010).
384
From all the discussion above, lipid vesicular systems including conventional liposomes, 385
invasomes (deformable liposomes) and ethosomes can act as penetration enhancers to improve the 386
skin drug delivery by their vesicle lipid bilayers or their additives such as ethanol and terpenes 387
interacting with the SC and subsequently modifying the SC intercellular lipid lamellae. However, 388
this penetration enhancing effect of lipid vesicular systems could play a much more important role 389
in the enhanced skin delivery of hydrophilic drug such as CF than in the case of lipophilic drug such 390
as mTHPC because this penetration enhancing effect just increases the partitioning of CF into the 391
lipophilic SC but does not really increase the partitioning of mTHPC from the SC into the less 392
lipophilic epidermis. Hence, for a lipophilic drug such as mTHPC, the entrapment of the drug in 393
vesicular lipid bilayers and intact vesicles penetration could be crucial for optimum skin deposition 394
and transdermal permeation. From this point of view, it is almost impossible for conventional 395
liposomes to reach this aim. For deformable liposomes such as invasomes and ethosomes, it is 396
possible for both of them to achieve this purpose because they somehow can act as drug carrier 397
systems, whereby intact vesicles enter the SC carrying vesicle-bound drug molecules into the skin.
398
However, for deformable liposomes such as invasomes, it should be pointed out that the driving 399
force for them entering the skin is xerophobia which is the tendency to avoid dry surroundings of 400
water-“loving” phospholipids (Cevc and Blume, 1992) and recent evidence showed that the water 401
gradient across the skin may not be linear and there may be a relatively ‘dry’ region within the 402
stratum corneum . It was also noticed that even in fully hydrated state, the water content in the 403
lowest stratum corneum layers close to the viable epidermis is much lower than in central regions of 404
the stratum corneum (Williams, 2003). Therefore, it was expected that, as a result of the osmotic 405
force, deformable liposomes will not penetrate beyond the level of the lowest layers in stratum 406
corneum. Regarding ethosomes, from the results of this study, it is also not very successful to deliver 407
mTHPC into deeper layers of skin. Therefore, other or better designed carrier systems for mTHPC 408
should be developed.
409
Another important technology which can also improve significantly the skin delivery for both 410
CF and mTHPC is the application of hydroethanolic solution (Table 1, CF-HE and mTHPC-HE).
411
From the results, in the case of CF, CF-HE significantly increased the CF accumulation in SC layers 412
(SC L1-5 and SC L6-10) compared with all other formulations containing CF except CF-ETS.
413
While, in the case of mTHPC, mTHPC-HE showed the highest mTHPC accumulation in both SC 414
layers and epidermis compared with all other formulation containing mTHPC. There are two 415
reasonable explanations responsible for its enhanced drug skin delivery effect. The first one is the 416
penetration enhancing effect by ethanol, which has been already described in detail above. The 417
second one is the increased thermodynamic activity of drugs due to the incorporation of ethanol or 418
water. In the case of CF, because it is hydrophilic and has a lower solubility in ethanol than in water, 419
the incorporation of ethanol in water will increase the thermodynamic activity of CF compared with 420
aqueous solution containing the same concentration of CF such as CF-PBS, CF-CL and CF-INS 421
Both CF-ETS and CF-HE could significantly (p<0.01) increase the CF accumulation in SC 422
compared to the other formulations. Moreover, because the synergistic penetration enhancing 423
effect between ethanol, lipid vesicles and the possible intact vesicle penetration mechanism of 424
ethosomes, CF-ETS also showed the highest CF accumulation in epidermis and dermis compared 425
with all others. In the case of mTHPC, the situation is different. Because mTHPC is highly 426
lipophilic and its low solubility in water, the incorporation of water in ethanol will increase its 427
thermodynamic activity compared with ethanol solution containing the same concentration of 428
mTHPC such as mTHPC-ET. For the lipid vesicular systems containing mTHPC in this study, even 429
though high water amounts are involved (see Table 1), entrapment of mTHPC in the lipid bilayers, 430
in fact, solubilizes mTHPC. Hence, the thermodynamic activity of mTHPC of these systems is not 431
equally increased compared with mTHPC-HE, which explains why mTHPC-HE showed the best 432
potential of improving mTHPC skin delivery.
433
3.3.2 Infinite dose application for CF and mTHPC in vitro study 434
Full-thickness human skin penetration and deposition data for application of an infinite dose 435
(160µL/cm2) for CF or mTHPC after 12 hr non-occlusive treatment with a range of formulation 436
vehicles are shown in Table 5 and Table 6, respectively, with their distribution in different skin 437
layers profiles shown in Figure 6 and Figure 7, respectively.
438
Table 5 should be inserted here 439
Table 6 should be inserted here 440
In the case of CF, the highest CF accumulation from all the test formulations (Table 5) was 441
found in the SC L1-5 where CF accumulation decreased in the following order: CF-ETS > CF-HE >>
442
CF-CL > CF-INS > CF-PBS. CF accumulation in SC L1-5 was significantly improved by CF-ETS 443
and CF-HE compared with CF-PBS (by a factor 33.9 and 17.9, respectively, p <0.01), while CF-CL 444
and CF-INS also significantly improved CF accumulation in SC L1-5 compared with CF-PBS, but 445
to a smaller extent (by a factor of 3.8 and 1.9, respectively, p <0.01). CF accumulation in SC L6-10
to a smaller extent (by a factor of 3.8 and 1.9, respectively, p <0.01). CF accumulation in SC L6-10